Gen-ichi Atsumi

CLOCK is involved in obesity‐induced disordered fibrinolysis in ob/ob mice by regulating PAI‐1 gene expression

Summary.  Background: An increased level of obesity‐induced plasma plasminogen activator inhibitor‐1 (PAI‐1) is considered a risk factor for cardiovascular disease. Aim: The present study investigates whether the circadian clock component CLOCK is involved in obesity‐induced PAI‐1 elevation. Methods: We examined plasma PAI‐1 and mRNA expression levels in tissues from leptin‐deficient obese and diabetic ob/ob mice lacking functional CLOCK protein. Results: Our results demonstrated that plasma PAI‐1 levels were augmented in a circadian manner in accordance with the mRNA expression levels in ob/ob mice. Surprisingly, a Clock mutation normalized the plasma PAI‐1 concentrations in accordance with the mRNA levels in the heart, lung and liver of ob/ob mice, but significantly increased PAI‐1 mRNA levels in adipose tissue by inducing adipocyte hypertrophy in ob/ob mice. The Clock mutation also normalized tissue PAI‐1 antigen levels in the liver but not in the adipose tissue of ob/ob mice. Conclusion: These observations suggest that CLOCK is involved in obesity‐induced disordered fibrinolysis by regulating PAI‐1 gene expression in a tissue‐dependent manner. Furthermore, it appears that obesity‐induced PAI‐1 production in adipose tissue is not closely related to systemic PAI‐1 increases in vivo.

Circadian clock molecules CLOCK and CRYs modulate fibrinolytic activity by regulating the PAI-1 gene expression

Summary.  Disruptions of circadian rhythms are associated with the development of many disorders. However, whether a disruption of the circadian clock can cause anomalies of the hemostatic balance remains unknown. The present study examines coagulation and fibrinolytic activities in circadian clock mutants, a homozygous Clock mutant and Cry1/Cry2 double knockout (Cry1/2‐deficient) mice. The euglobulin clot lysis time (ELT) showed circadian variations that peaked at 21:00 (early night) in wild‐type mice, suggesting that fibrinolytic activity is lowest at this time. The ELT was continuously reduced in Clock mutants, while the ELT was significantly increased and did not differ between day and night (9:00 and 21:00) in Cry1/2‐deficient mice. The prothrombin time (PT) and activated partial prothrombin time (APTT) were constant in all genotypes. To identify which factors cause the loss of ELT rhythm, we measured fibrinolytic parameters in Clock mutant and Cry1/2‐deficient mice. The robust circadian fluctuation of plasma plasminogen activator inhibitor 1 (PAI‐1) that peaked at early night was damped to trough levels in Clock mutant mice. On the other hand, PAI‐1 levels in Cry1/2‐deficient mice remained equivalent to the peak levels of those in wild‐type mice at both 9:00 and 21:00. Circadian changes in plasma PAI‐1 levels seemed to be regulated at the level of gene expression, because the plasma PAI‐1 levels in Clock mutant and Cry1/2‐deficient mice were closely correlated with the level of PAI‐1 mRNA transcript in these mice. Plasma plasminogen and hepatic mRNA levels were not rhythmic in wild‐type mice, and continuously higher in Clock mutant than in wild‐type or Cry1/2‐deficient mice. In contrast, the activity and mRNA levels of tissue type plasminogen activator (t‐PA), plasma levels and mRNA levels of plasminogen, and plasma levels of α2 plasmin inhibitor (α2PI) in all genotypes were constant throughout the day. Coagulation parameters such as factor VII, factor X, prothrombin and fibrinogen remained constant throughout the day, and were not affected by clock gene mutations. These results suggest that circadian clock molecules play an important role in hemostatic balance by regulating the fibrinolytic systems.

Circadian variations in coagulation and fibrinolytic factors among four different strains of mice.

This study examined circadian variation in coagulation and fibrinolytic parameters among Jcl:ICR, C3H/HeN, BALB/cA, and C57BL/6J strains of mice. Plasma plasminogen activator inhibitor 1 (PAI‐1) levels fluctuated in a circadian manner and peaked in accordance with the mRNA levels at the start of the active phase in all strains. Fibrinogen mRNA levels peaked at the start of rest periods in all strains, although plasma fibrinogen levels remained constant. Strain differences in plasma antithrombin (AT) activity and protein C (PC) levels were then identified. Plasma AT activity was circadian rhythmic only in Jcl:ICR, but not in other strains, although the mRNA levels remained constant in all strains. Levels of plasma PC and its mRNA fluctuated in a circadian manner only in Jcl:ICR mice, whereas those of plasma prothrombin, factor X, factor VII, prothrombin time (PT), and activated partial thrombin time (APTT) remained constant in all strains. These results suggest that genetic heterogeneity underlies phenotypic variations in the circadian rhythmicity of blood coagulation and fibrinolysis. The circadian onset of thrombotic events might be due in part to the rhythmic gene expression of coagulation and fibrinolytic factors. The present study provides fundamental information about mouse strains that will help to understand the circadian variation in blood coagulation and fibrinolysis.

Xanthoangelols isolated from Angelica keiskei inhibit inflammatory-induced plasminogen activator inhibitor 1 (PAI-1) production.

The folk medicine Angelica keiskei (Ashitaba) exhibits antitumor, antioxidant and antidiabetic activities and it has recently attracted attention as a health food. Ashitaba is thought to have antithrombotic properties, but this has not yet been scientifically proven. The elevation of plasma plasminogen activator inhibitor 1 (PAI-1), an inhibitor of fibrinolysis results in a predisposition to the risk of thrombosis. The present study showed that Ashitaba exudates injected intraperitoneally and orally administered over long-term suppressed the lipopolysaccharide (LPS) induced PAI-1 increase in mouse plasma. We also found that xanthoangelol, xanthoangelols B and D, the components of Ashitaba exudates, significantly inhibited TNFα-induced PAI-1 production from human umbilical vein endothelial cells (HUVECs). These findings suggest that Ashitaba can decrease elevated PAI-1 production, and that daily consumption of Ashitaba product might maintain anticoagulant status by inhibiting elevations in PAI-1 under inflammatory conditions.

Experimental study on the hemostatc activity of Pollen Typhae: a traditional folk medicine used by external and oral application.

Pollen Typhae is the traditional Chinese herbal medicine widely used to treat the hemorrhagic diseases both by external and oral application. The present study examines the hemostatic properties and its components of Pollen Typhae. Pollen extract significantly reduced prothrombin time (PT), activated partial prothrombin time (APTT) and recalcification time. Pollen extract directly activated factor XII in the coagulation cascade. Acidic polysaccharide in the pollen that adsorbed to the diethylaminoethyl (DEAE) column was the causative agent of factor XII activation. These results suggested that an electronegative charge attributed to an acidic polysaccharide in the pollen extract contributed to the hemostatic activity. We then examined the hemostatic activity of administered pollen extract in the mouse tail bleeding model. Tail bleeding was significantly decreased after oral administration of the pollen extract, whereas the acidic polysaccharide fraction did not affect the duration of tail bleeding. These results suggest that the oral anticoagulant effect of Pollen Typhae is attributed to compounds other than acidic polysaccharides. We concluded that the activation of the intrinsic coagulation pathway by the acidic polysaccharide contributes to the external hemostatic property of Pollen Typhae, and the action of components such as flavonoids that possess anticoagulant activity are causative agent when orally administered.

Blood coagulation and metabolic profiles in middle-aged male and female ob/ob mice.

Obese and diabetic states in humans are associated with an increased incidence of thrombotic diseases caused by various coagulation abnormalities. Genetically obese ob/ob mice produce metabolic abnormalities similar to those associated with type 2 diabetes. However, little is known about their coagulation features or sex differences. The present study aimed to determine the effects of obese and diabetic complications on blood coagulation and vascular diseases by exploring correlations between blood coagulation and metabolic profiles in middle-aged male and female ob/ob mice. Plasma levels of plasminogen activator inhibitor 1 (PAI-1) were significantly increased, whereas those that of platelet factor-4 (PF-4) was slightly, but significantly increased in male and female ob/ob mice compared with lean counterparts. Prothrombin time (PT) was significantly shortened in female ob/ob mice and activated partial thrombin time (APTT) significantly differed between male and female ob/ob mice. Plasma levels of antithrombin (AT) were significantly increased in male and female ob/ob mice. None of the other coagulation and fibrinolytic factors examined significantly differed between ob/ob mice and lean counterparts. On the contrary, factors such as body weight and cholesterol levels significantly differed between ob/ob and lean mice, whereas glucose, fructosamine and insulin levels significantly differed only in one sex of each strain. These results provided fundamental information about blood coagulation and metabolic features for exploring the function of altered blood coagulation states in ob/ob mice.

Differentiation with elaidate tends to impair insulin-dependent glucose uptake and GLUT4 translocation in 3T3-L1 adipocytes

Abstract Development of type 2 diabetes mellitus and insulin resistance is associated with a quality of dietary fatty acids such as saturated and unsaturated fatty acids. Dietary fatty acids also include transform of unsaturated fatty acids and intake of transform of oleate (elaidate) is associated with cardiovascular disease. However, little is known about the roles of elaidate in insulin responsiveness. We show here that elaidate impairs insulin-dependent glucose uptake in adipocytes. Differentiation with 10 μM elaidate, which is close to physiological plasma concentration, reduces insulin-dependent glucose uptake. Furthermore, insulin-dependent GLUT4 translocation is disturbed in adipocytes differentiated with elaidate. In addition, analysis of lipolysis and gene expression shows that deteriorative effects of elaidate on insulin responsiveness are limited but not general. Thus, our findings reveal that differentiation with elaidate tends to affect insulin-dependent glucose uptake through alternation of GLUT4 translocation from cytosol to the plasma membrane.

Screening for hemostatic activities of popular Chinese medicinal herbs in vitro.

Aims: This study aimed to identify new hemostyptics by assessing the coagulation enhancing activity of 114 Chinese herbal extracts in vitro. Methods: Herbs were boiled in water for 30 min, filtered and then lyophilized filtrates (10 mg/mL) were dissolved in water. Coagulation was assayed as prothrombin time (PT). Plasma diluted in saline was incubated with each extract for 5 min and then PT reagent was added, followed by CaCl2 solution and the time taken to form clots was measured. Extracts that decreased coagulation time were regarded as containing active compounds. The abilities of extracts to activate Factor XII were assessed and the activated form of factor XII (XIIa) was resolved by SDS-PAGE and visualized by silver staining. Results: Coagulation time was obviously shortened by extracts of Alpinia Rhizome, Areca, Artemisia Leaf, Cassia Bark, Danshen Root, Ephedra Herb, Epimedium Herb, Forsythia Fruit, Great Burdock Achene, Moutan Bark, Perilla Herb, Red Paeony Root, Schizonepeta Spike, Senticosus Rhizome, Sweet Annie, Uncaria Thorn and Zanthoxylum Peel. Factor XII was obviously activated by extracts of Artemisia Leaf and Great Burdock Achene, and slightly by Perilla herb. Conclusion: Some popular Chinese medicinal herbs have potential as hemostatic agents and could thus be develope as new strategies for the treatment and prevention of bleeding.

Activation of PPARγ at an Early Stage of Differentiation Enhances Adipocyte Differentiation of MEFs Derived from Type II Diabetic TSOD Mice and Alters Lipid Droplet Morphology

Type 2 diabetic Tsumura, Suzuki, obese, diabetes (TSOD) mice gradually gain weight as compared to corresponding Tsumura, Suzuki, non-obesity (TSNO) control mice, and develop insulin resistance. Although development of type 2 diabetes mellitus is associated with dysfunction of adipocytes, little is known about the properties of adipocytes from TSOD mice. Therefore, we attempted to remove intracorporeal factors and elucidate inherent properties of adipocytes of TSOD mice using adipocytes differentiated from mouse embryonic fibroblasts (MEFs) in vitro. Here, we show that MEFs of TSOD have low potency for differentiation into adipocytes. The percentage of Oil red O-stained cells and levels of adipogenic markers in cells differentiated from MEFs of TSOD are lower than those in cells differentiated from MEFs of TSNO. We further show that treatment with an agonist of peroxisome proliferator-activated receptor-γ (PPARγ) (rosiglitazone) at an early stage of differentiation increases the percentage of Oil red O-stained cells in TSOD-MEFs differentiated into adipocytes. Moreover, the lipid droplet size in those adipocytes is larger than that in the adipocytes differentiated from MEFs of TSNO. Although persistent treatment of MEFs of TSOD with rosiglitazone during differentiation increases the percentage of Oil red O-stained cells, the lipid droplet size in adipocytes treated as such does not reach the size of those treated in early stage only. Thus, activation of PPARγ by its agonist at an early stage of differentiation compensates for the low potency toward adipogenic differentiation of, and accelerates formation of enlarged lipid droplets in adipocytes derived from, MEFs of TSOD mice.

Effects of a diet containing Brazilian propolis on lipopolysaccharide-induced increases in plasma plasminogen activator inhibitor 1 (PAI-1) levels in mice -

Background: Brazilian propolis has many biological activities including the ability to help prevent thrombotic diseases, but this particular effect has not been proven. Plasma levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, increase under inflammatory conditions such as infection, obesity and atherosclerosis and such elevated levels predispose individuals to a risk of developing thrombotic diseases. Aim: This study aimed to determine the effects of a diet containing Brazilian propolis on lipopolysaccharide (LPS)-induced increases in plasma PAI-1 levels. Materials and Methods: Mice were fed with a diet containing 0.5% (w/w) Brazilian propolis for 8 weeks. Thereafter, the mice were subcutaneously injected with saline containing 0.015 mg/kg of LPS and sacrificed 4 h later. Results: Orally administered Brazilian propolis significantly suppressed the LPS-induced increase in PAI-1 antigen and its activity in mouse plasma. Conclusion: This study indicated that Brazilian propolis contains natural products that can decrease thrombotic tendencies in mice.